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Intercal - Advanced molecules, membranes and cells > Patch Clamp Techniques 1&2 > Flashcards

Patch Clamp Techniques 1&2 Flashcards

1
Q

What is resistance

A

a measure of the opposition to current flow in an electrical circuit.

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2
Q

What is current

A

Rate of flow of charged particles aka the charge transfered over time

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3
Q

What is voltage

A

The electrical potential difference - no current if voltage the same at both ends to need a difference between the ends.
E.g. 10V at one end 0V at the other, will flow towards 0V

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4
Q

What is ground

A

A point of reference - no change in voltage but different starting point e.g. 20mV instead of 0 so inside the cell was 60mV but with ground included its 40mV

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5
Q

What is the relationship between current resistance and flow

A

All rely on each other so -
V=IR

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6
Q

What is capacitance

A

The change in potential (charge)

Units - farads

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7
Q

What is the role of a capacitors

A

A capacitor stores and releases current in a circuit - can release current when no voltage if the energy is stored

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8
Q

What has capacitance in cells

A

Cell membrane

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9
Q

What has current in cells

A

Ions flowing through cell membrane

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10
Q

What has voltage in cells

A

The potential difference across the membrane

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11
Q

What has resistance in cells

A

Ion channels

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12
Q

Does the number of channels (resistors) in parallel effect the current

A

No - current spread over 1 or 20 resistors, it doesnt matter, all add up to total current

It = I1 + I2 + I3 etc

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13
Q

What is the relationship with conductance and resistance

A

Conductance is the inverse of resistance

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14
Q

What happens at the ion equilibrium potential

A

There is no net flow of ions across the membrane - it is different for different ions

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15
Q

What two factors allows the cell to have a membrane potential

A

Concentration gradient is due to ion pumps (Na/K ATPase)

Electrical potential is due to selective membrane permeability

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16
Q

What is the resting potential of neuron cells

A
  • 60mV
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17
Q

What is the resting potential of muscle cells

A
  • 90mV
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18
Q

Where is there more potassium

A

Inside the cell

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19
Q

Where is there more sodium

A

Outside the cell

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20
Q

Where is there more chloride

A

Outside the cell

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21
Q

Where is there more calcium

A

Outside the cell

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22
Q

What is the purpose of the voltage clamp

A

Allows the voltage across the membrane to be fixed and this allows the current flowing across that membrane to be measured.

This allowed analysis of single ion channels as allowed control of the external and internal cell environments and recorded low noise (single-channel currents)

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23
Q

How does the patch clamp technique work

A

Isolates a patch of membrane (pinches) from the external solution with a glass pipette and record current flowing through the patch (a single channels current)

24
Q

What is important about the patch seal resistance

A

Needs to be tight. Therefore the seal resistance must be greater than the pipette to stop current flowing outwith patch as the true isolated current will not be obtained if allowed to escape.

25
Q

What does a loose seal give

A

Escaped current
AND
The background noise

26
Q

What is a gigaseal

A

Mechanically stable high resistance seal which ensures as much current as possible enters the glass pipette

27
Q

How are gigaseals made

A

Suction to the membrane of the cell increases the seal - gives gigaseal

Allows even the smallest of currents to be obtained

28
Q

Give the 4 types of patch-clamp gigaseal

A

Cell attached - suction of patch but patch still attached to cell

Inside out patch - suction of patch so much that it tears away from cell that the inside of the cell is present in the glass pipette

Whole cell recording - suction of patch is removed and make recordings for the whole of the inside of a cell

Outside-out patch - suction of patch so much that it tears away from cell that the outside is present in the glass pipette

29
Q

What was found with ion channels using patch clamp techniques

A

Clamps are always opening and closing
Its instantaneous
Modulators

30
Q

What are the advantages of using cells for patch clamps

A

Easier access
Can be used to express specific proteins and their effects
Better space clamp - amplifier can apply the voltage quite well across the membrane unlike the complexity of tissue and multiple cells which is harder

31
Q

What are the advantages of using tissue sections for patch clamps

A

Connectivity is preserved
No culture media - so no added solutions which may not be physiological

32
Q

What is the main purpose of the solution the cells are in

A

Mimics the composition of the fluid in vivo (extracellular and pipette solution - intracellular)

33
Q

What is the purpose of a vibratome

A

Has a vibrating blade and tissue holder to cut the tissue sections

34
Q

What is the purpose of a pipette puller

A

Has a heating element and glass capillary holder.
This heats the glass and produces a narrow tip which is the pipette.

35
Q

What is the purpose of the amplifier

A

Maintains a constant membrane potential and is attached to the pipette

36
Q

How do you know that you are clamping the right area

A

Has pipette holder with a micromanipulator and microscope to specifically target the correct channel

37
Q

How does patching occur

A

Clear flow of liquid outside of the pipette with air to allow clean contact with the cell membrane.
Can measure current and knows the voltage we are applying using amplifier so we can tell with V=IR the resistance of the pipette to see if the pipette is sufficient (Gigapipette)

38
Q

How do we know if the micropipette is touching the cell

A

Using V=IR –>

Know constant voltage by amplifier
Decrease in current and increase in resistance shows that it is attached.

39
Q

What happens to current when patch suction occurs

A

Current decreases from very high to tiny as removal of background noise and left with single channel current. Also shows that voltage is very high (desired as gigapipette)

40
Q

How does whole cell recording work

A

More section occurs until the cell membrane breaks, this causes a drop in current which is then compensated which shows the whole cell current

41
Q

What is the difference between voltage clamps and current clamps

A

Voltage clamp - set voltage, measures current (e.g. post-synaptic currents)

Current clamp - set current, measures voltage
(e.g. action potentials)

42
Q

Why are cell-attached recordings important

A

Can look at changes across the membrane without disturbing the whole cell

43
Q

Can whole cell patch clamp be done in vivo

A

Yes and vitro

44
Q

How can patch clamp help in pharmacology

A

Tool to investigate cell mechanism using well studied drug
Tool to investigate drug mechanism using well studied cell

45
Q

How can whole cell recordings be used in post-synaptic currents

A

Patch on input cell for whole cell recording and the currents can be recorded.
Stimulus - specific stimulus to pre-synaptic neuron and see its effects in post-synaptic cell
Known inhibitor of pre-synaptic neuron and see its effects on post-synaptic cell (see spontaneous actions of post-synaptic neuron independent of pre-synaptic)
Use drugs to block channels and assess its effects

46
Q

How does patch clamp techniques and optogentics work together

A

Channelrhodopsin (light sensitive cation channel) opens to blue light.
Current can be recorded via optogenetics in the channelrhodopsin neuron or its post-synaptic neuron (channelrhodopsin-assisted circuit mapping - CRACM)

47
Q

How do automated patch clamp systems work

WIDER READING - Vasilyev et al 2005

A

Collects large amounts of data quickly over a shorter time than traditional patch clamp techniques.

This technique is when cells are placed on an injection molded cast chip which cells attach to. This substitutes the micropipette and has an integraded electrode for controlling voltage or current.

This allows cells to be inside out or whole cell patch clamp recordings.

This allows different solution wells to be applied to the cells to look at their reactions.

48
Q

What is electrode fabrication and why is it important

WIDER READING - (Kornreich, 2007)

A

The formation of the micropipette by melting it with an electrode.
To ensure a tight seal with no impurities - all solutions are flitered via a micronfilter before experiments and electrode fabrication of micropipettes are done as close as possible to the time they are being used and then kept in sealed chamber to prevent airborne particles from contaminating the tips.

49
Q

What kind of glass is used for the micropipette and why

WIDER READING - (Kornreich, 2007)

A

Borosilicate glass - low softening temp to make the pipette, low electrical noise, forms high resistance seals easily.

50
Q

What is two-photon targeted patching (TPTP) and what did it discover in parvalbumin-EGFP positive interneurons in the somatosensory cortex.

WIDER READING - Margrie et al 2003

A

Uses two-photon imaging (imaging used for dense tissue with penetration of 1mm) to guide in vivo whole-cell recordings of labelled cortical parvalbumin-EGFP positive interneurons in the somatosensory cortex.

Showed that spontaneous and evoked spikelets (spike depolarisations) are needed to be synchronized to terminate episodes of strong neuronal network excitation.

51
Q

What is an advantage of the cell-attached current clamp over whole-cell recording

WIDE READING - Perkins 2006

A

An advantage of cell-attached current-clamp over whole-cell recording is that it accurately depicts whether a synaptic potential is hyperpolarizing or depolarizing without the risk of changing its polarity.

52
Q

What was obtained regarding cardiomyocytes and patch-clamp techniques?

WIDER READING - Verkerk et al 2021

A

Hard to test drugs on cardiomyocytes as frequently depolarized state when isolated.
Did a dynamic clamp of providing an inward rectifier K current to form a non-depolarized resting membrane potential.
This allowed the testing of multiple drugs - lidoaine, nifedipine etc) which evoked APs so showed it worked.
Therefore, dynamic clamp represents a promising tool for testing novel antiarrhythmic drugs.

53
Q

Why are sperm difficult to test patch-clamp techniques on

WIDER READING - Lishkko et al 2014

A

Tight, small cell membrane with lack of cytoplasm so hard to get gigaseal with micropipette.
However, found a region on the plasma membrane called the cytoplasmic droplet that is loosely attached to the rigid intracellular structures and with which a tight gigaohm seal can be formed with the patch pipette

54
Q

Using patch-clamp testing, what was discovered about sperm motility

WIDER READING - Kirichok et al 2006

A

Calcium and alkali dependent - Sperm flagella become hyperactivated due to an influx of calcium via CatSper1 channel because of intracellular alkalization (discovered via patch clamp) which causes the hypermobility.

55
Q

Using a whole-cell patch-clamp, what was found regarding malaria and its effects on RBCs

WIDER READING - Bouyer et al 2007

A

Did whole-cell patch clamp testing on RBCs infected with malaria parasite plasmodium falciparum in physiological ion concentrations.

Shown that the membrane current is 100-150 fold higher than in uninfected RBCs compared to infected cells.

This was because - the inwardly rectifying anion channel and Ps small conductance anion channel have enhanced activity in these infected cells. Shown that erythrocyte permeability is increased by the parasite. Proven with whole cell patch clamp.

56
Q

What did patch clamp techniques show regarding diclofenac in mice airways

WIDER READING - Chen et al 2019

A

Diclofenac relaxes the smooth muscle in the tracheal by relaxing high potassium and Ach contraction of the tracheal rings via decreasing ctosolic calcium in the airway smooth muscle cells through inhibiting voltage gated calcium channels preventing calcium influx which prevents contraciton.

Suggests - diclofenac may be good as a antibronchospasmic drug used in treating respiratory diseases such as asthma and chronic obstructive pulmonary disease.

Intercal - Advanced molecules, membranes and cells (31 decks)

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